GFP-LC3 High-content Assay for Screening Autophagy Modulators

Abstract

Autophagy plays an important role in maintaining cellular homeostasis. Defects in autophagy have been linked to various human diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Therefore, it is useful to develop an assay that can measure the functions of autophagy and be also used to identify autophagy modulators by screening a large number of compounds. This chapter describes a cell-based high content Green Fluorescent Protein (GFP)-LC3 assay using mouse embryonic fibroblasts (MEF) stably expressing GFP-LC3.

1. Introduction

Autophagy, a major intracellular degradation system, removes unnecessary or dysfunctional components [1] to allow the orderly degradation and recycling of cellular components [2,3]. It has become increasingly clear that autophagy plays an important role in homeostasis. Defects in autophagy have been linked to various diseases, including neurodegeneration and cancer. Increased autophagy has been shown to delay aging, improve neuronal functions and protect against tissue injury. Therefore, interest in modulating autophagy as potential therapeutic targets for these diseases has grown rapidly [4–6].

Microtubule-associated protein 1 light chain 3 (LC3), a ubiquitin-like protein that is important for the formation of the autophagosome, is a reliable marker to monitor autophagy. The behavior of GFP-LC3 fusion protein is very similar to the endogenous LC3, which has been widely used as a marker to monitor autophagy [7,8]. An increased number of GFP-LC3 puncta can also be due to the impaired fusion of autophagosomes with lysosomes or impaired lysosomal functions.

A GFP-LC3 stable cell line used to measure autophagic flux has been previously developed and optimized in a 1536 well plate format, and then validated by screening the LOPAC library [9]. Recently, this assay has been applied to evaluate a group of anti-SARS-CoV-2 compounds. Many of these anti-SARS-CoV-2 compounds were identified as autophagy modulators using this assay [10]. This assay takes advantage of the distinctive pattern of cellular GFP-LC3 upon autophagy induction. Under nutrient-rich conditions, the level of autophagy is low and GFP-LC3 displays a diffuse pattern. However, during cellular stress, the process of intracellular autophagy is increased. GFP-LC3 conjugates with phosphatidylethanolamine (PE) and the PE-conjugated GFP-LC3 translocates to the autophagosomal membrane and displays a punctate pattern. The number of GFP-LC3 puncta per cell can be quantified and represents the number of autophagosomes in each cell. We used chloroquine diphosphate, which increases lysosomal pH and blocks lysosomal degradation, as a positive control. This chapter describes the detailed GFP-LC3 cell-based high-content assay using an automated high content screening system.

2. Materials

2.1. Equipment

1. Purifier Logic+ Class II, Type A2 Biosafety Cabinet for cell operations.

2. Steri-Cult CO2 Incubator for culturing cells at 37 °C under a humidified atmosphere and 5% CO2.

3. Eight-tip multidrop reagent dispenser (Thermo Fisher Scientific, Waltham, MA).

4. BioRAPTR Flying Reagent Dispenser (FRD, Beckman Coulter Inc., Brea, CA).

5. Pin tool station (Wako Automation, San Diego, CA).

6. BlueWasher (Blue Cat Bio, Concord, MA).

7. Operatta CLS High Content Screening System with software of Harmony 4.6. (PerkinElmer, Waltham, MA).

2.2. Reagents/Supplies

1. Mouse embryonic fibroblast (MEF)-GFP-LC3 cells were stable transfected with GFP-LC3 plasmid from Wen-Xing Ding’s lab [9]. Culture medium (CM): DMEM + GlutaMax (Invitrogen) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT) and 50 U/ml penicillin and 50μg/ml streptomycin (Invitrogen, CA).

2. Positive control compounds: chloroquine diphosphate (CQ) (Sigma-Aldrich).

3. Collagen I-coated 1536-well black wall/clear bottom plate (Corning, Acton, MA).

4. Hoechst 33342 (Molecular Probes).

5. Fixing solution: 8% (v/v) Paraformaldehyde (from 32% stock) and 1 μg/mL Hoechst 33342 (from 10 mg/ml stock) in DPBS. Prepare it before use.

6. TopSeal-A: Clear self-adhesive topseal for 384-well Microplate (PerkinElmer, Waltham, MA).

7. Corning^™ Sterile Cell Strainers (Corning^™ 431750, Fisher Scientific).

3. Method

3.1. Thawing cells

1. Add 9 mL of pre-warmed cell culture DMEM medium supplemented with 10% fetal bovine serum, penicillin and streptomycin, and glutamine in a 15 mL conical tube.

2. Thaw a vial of MEF-GFP-LC3 cells in a 37 °C water bath for 1–2 min.

3. Transfer thawed cells to the conical tube and gently mix the contents.

4. Centrifuge the tube for 4 min at 200 × g at room temperature.

5. Aspirate supernatant and resuspend cell pellet with 10 mL of cell culture medium.

6. Count the total cell number and transfer 2 × 10⁶ to a T75 flask with a final medium volume of 10 mL.

7. Incubate the flask in a humidified incubator at 37°C, 5 % CO₂ until 80 % confluence.

3.2. Culturing Cells

1. Aspirate cell culture medium from a T75 flask of cells.

2. Rinse cell layer with calcium /magnesium-free DPBS.

3. Add 2 mL of 0.05 % Trypsin–EDTA to the flask.

4. Incubate the flask in a humidified incubator at 37 °C, 5 % CO₂ until all cells are detached as verified by a tissue culture microscope.

5. Add 4 mL of cell culture medium to the flask to terminate Trypsin-EDTA action.

6. Transfer the detached cells to a 50 mL conical tube.

7. Centrifuge the tube for 4 min at 200 × g at room temperature.

8. Aspirate supernatant and resuspend the cell pellet with 10 mL of cell culture medium.

9. Count total cell number and passage cells at 1:10 – 1:15 ratios twice per week.

10. Incubate the flask in a humidified incubator at 37°C, 5 % CO₂ until 70–80 % confluence.

3.3. Plating Cells

1. Aspirate cell culture medium from a flask of cells.

2. Rinse cell layer with calcium /magnesium-free DPBS.

3. Add 2 mL per T75 flask or 5 mL per T225 flask of 0.05 % Trypsin–EDTA to flask.

4. Incubate the flask in a humidified incubator at 37 °C, 5 % CO₂ until all cells are detached as verified by a tissue culture microscope.

5. Add 4 mL of pre-warmed cell culture medium to the T75 flask or 10 mL per T225 flask to terminate Trypsin-EDTA action.

6. Transfer the detached cells to a 50 mL conical tube.

7. Centrifuge the tube for 4 min at 200 × g at room temperature.

8. Aspirate supernatant and resuspend the cell pellet with 10 mL of culture medium.

9. Pass the cell suspension through a cell strainer and collect the flow through (see Note 1).

10. Count the cell number and dilute to 1.6 × 10⁵ cells per mL in culture medium.

11. Dispense 5 μL of 800 cells to each well (i.e., 800cells/well/5μL) in a Collagen I-coated 1536-well black clear bottom plate (see Note 2) using BioRAPTR flying reagent dispenser or an 8-tip multidrop reagent dispenser.

12. Place a porous metal lid on top of the assay plate and incubate in a humidified incubator at 37°C, 5 % CO₂ for 6 hours for cells to adhere.

3.4. Compound Treatment

1. After 6 hours of incubation, add 23 nL of chloroquine diphosphate (positive control) or DMSO (negative control) from the control plates (Fig. 1) to the assay plate using Wako Pintool Station.

2. Incubate assay plates at 37°C/5% CO2 for 18 hours.

Fig. 1.

Control compound plate map in a 1536-well plate format.

3.5. Fixation and Staining

1. Fix the cells by adding 5μL/well of freshly made fixing solution containing 8% (v/v) paraformaldehyde (final concentration at 4%) and 1μg/ml Hoechst 33342 (final concentration at 0.5 μg/ml) on top of the assay wells (see Note 3) using BioRaptr.

2. Incubate plates at room temperature for 30 min, remove fixing solution and wash with HBSS (8μl/well) twice using gentle spin function of BlueWasher (see Note 4), and then add 5 μL of HBSS/well.

3. Seal the assay plates for imaging or store at 4 °C for future imaging.

3.6. Image Readout

The assay plates were imaged via Perkin Elmer Operatta CLS using 20x objective, confocal EGFP channel (Excitation 460–490 nm, Emission 500–550 nm) and DAPI (Excitation 355–385 nm, Emission 430–500 nm), which measured fluorescence intensity. Images were acquired for one center field (around 25% of a single well area in a 1536-well plate) in each well. Adjusting the exposure time, power, and height for each channel optimized image clarity.

3.7. Image Analysis

Images were analyzed with Operetta Harmony 4.6 software. The total number of nuclei in a well represents the number of cells in the well. The compartment analysis algorithm was used to identify the nuclei, apply a cytoplasmic mask, and quantitate GFP spots in the GFP channel. A nuclear mask was generated from DAPI stained nuclei. Autophagosomal membrane-associated GFP-LC3 (puncta) was detected as GFP-fluorescent vesicular objects that exceeded a threshold defined by untreated cells and found exclusively in the cytoplasmic area. Data was expressed as four output parameters: % of LC3 positive cells, Number of Spots - Mean per Well, Total Spot Area - Mean per Well, and Relative Spot Intensity - Mean per Well. The positive control of inducing autophagy, chloroquine diphosphate, showed clear concentration dependency in this assay (Fig. 2). The compound half-maximum effective concentration (EC₅₀) and maximum response (efficacy) values were calculated using a four-parameter Hill equation in GraphPad Prism software (Fig. 3). The assay performance is showed in table 1.

Fig. 2.

Induction of GFP-LC3 puncta by chloroquine diphosphate treatment in MEF-GFP-LC3 cells. Representative images are nuclei (blue) with GFP-LC3 puncta (green). Chloroquine diphosphate treatment for 18 h at 0, 18, and 27 μM, respectively.

Fig. 3.

Chloroquine diphosphate (CQ) induced GFP-LC3 in a concentration-dependent manner. EC₅₀ of CQ is 19.5 μM in MEF-GFP-LC3 cells in a 1536-well plate format.

Table 1.

Assay performance

% LC3 positive cells	Mean ± SD
CV (%)	29.72 ± 1.83
S/B	5.94 ± 0.46
Z’ factor	0.31 ± 0.07

Each value represents the mean ± SD of three independent experiments. CQ (20 μM) used as positive control and DMSO as negative control in the GFP-LC3 puncta formation

4. Note

1. Getting a single cell suspension is very critical for this high content assay for clear image and data analysis.

2. Coated plates are employed in this assay to eliminate the loss of cells during the washing steps.

3. Adding fix solution on top can simplify steps and minimize the loss of cells through removing medium before fixation.

4. Use the gentle spin function of BlueWasher to avoid cell loss during washing steps.